Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages

Sphingomyelinases secreted by pathogenic bacteria play important roles in host–pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface‐exposed C‐terminal sphingomyelinase domain and a putative N‐terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sp hingomyelinase of M ycobacterium t uberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtb's hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5‐ and 100‐fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth.     

Auxotrophy and intrapopulation complementary in the ‘interactome’ of a cultivated freshwater model community

Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet‐uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high‐quality metagenome‐assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin‐ and amino acid biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage‐based partitioning of the genes recovered from a single MAG intrapopulation metabolic complementarity was revealed pointing to ‘social’ interactions for the common good of populations dominating freshwater plankton. As such, our MAGs highlight the power of mixed cultures to extract naturally occurring ‘interactomes’ and to overcome our inability to isolate and grow the microbes dominating in nature.     

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na⁺ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na⁺ (Na⁺ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na⁺ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na⁺ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na⁺ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na⁺ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na⁺ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.     

Constraining local structure can speed up folding by promoting structural polarization of the folding pathway

The pathway which proteins take to fold can be influenced from the earliest events of structure formation. In this light, it was both predicted and confirmed that increasing the stiffness of a beta hairpin turn decreased the size of the transition state ensemble (TSE), while increasing the folding rate. Thus, there appears to be a relationship between conformationally restricting the TSE and increasing the folding rate, at least for beta hairpin turns. In this study, we hypothesize that the enormous sampling necessary to fold even two-state folding proteins in silico could be reduced if local structure constraints were used to restrict structural heterogeneity by polarizing folding pathways or forcing folding into preferred routes. Using a Gō model, we fold Chymotrypsin Inhibitor 2 (CI-2) and the src SH3 domain after constraining local sequence windows to their native structure by rigid body dynamics (RBD). Trajectories were monitored for any changes to the folding pathway and differences in the kinetics compared with unconstrained simulations. Constraining local structure decreases folding time two-fold for 41% of src SH3 windows and 45% of CI-2 windows. For both proteins, folding times are never significantly increased after constraining any window. Structural polarization of the folding pathway appears to explain these rate increases. Folding rate enhancements are consistent with the goal to reduce sampling time necessary to reach native structures during folding simulations. As anticipated, not all constrained windows showed an equal decrease in folding time. We conclude by analyzing these differences and explain why RBD may be the preferred way to constrain structure.     

GUN4-porphyrin complexes bind the ChlH/GUN5 subunit of Mg-Chelatase and promote chlorophyll biosynthesis in Arabidopsis

The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranes—the site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O₂. To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding–deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mg-chelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex.     

Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells

Background

Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.

Methodology

Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.

Results

Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.

Conclusion

Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.     

Growth and multiplication ofRhodotorula glutinis as determined by viable and total cell counts

Excellent correlation has been shown between two total and two culture, or viable, count techniques used for cell enumeration studies ofRhodotorula glutinis. The culture methods examined, the drop plate and the spread plate proved equally reliable for enumeration except for slight variations during the period of maximal budding of the organism. The Coulter Counter was a valuable tool as it permitted rapid cell population analysis with minimal preparatory procedures. The Petroff-Hausser counting chamber was found applicable only when cell numbers approached 10⁹ or greater.Contribution No. 468 from the Marine Laboratory, Institute of Marine Science, University of Miami, Miami, Florida, U.S.A. Supported by Grant G-16146 from the National Science Foundation.